U.S. patent application number 11/459626 was filed with the patent office on 2007-08-16 for solid phase synthesis.
This patent application is currently assigned to POSTECH Foundation. Invention is credited to Bong-Jin Hong, Joon-Won PARK.
Application Number | 20070190537 11/459626 |
Document ID | / |
Family ID | 38369028 |
Filed Date | 2007-08-16 |
United States Patent
Application |
20070190537 |
Kind Code |
A1 |
PARK; Joon-Won ; et
al. |
August 16, 2007 |
SOLID PHASE SYNTHESIS
Abstract
The present invention relates to a method for synthesis of a
polynucleotide on a dendron-modified surface of a substrate, and a
method for stably maintaining a polynucleotide immobilized on a
solid surface.
Inventors: |
PARK; Joon-Won;
(Pohang-city, KR) ; Hong; Bong-Jin; (Pohang-city,
KR) |
Correspondence
Address: |
JHK LAW
P.O. BOX 1078
LA CANADA
CA
91012-1078
US
|
Assignee: |
POSTECH Foundation
Pohang-city
KR
POSCO
Pohang-shi
KR
|
Family ID: |
38369028 |
Appl. No.: |
11/459626 |
Filed: |
July 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60701848 |
Jul 22, 2005 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
525/54.2; 536/24.3; 977/754; 977/924 |
Current CPC
Class: |
C12Q 1/6837 20130101;
B01J 2219/00626 20130101; B01J 2219/00677 20130101; B01J 19/0046
20130101; B01J 2219/00612 20130101; B01J 2219/00387 20130101; B01J
2219/00659 20130101; B01J 2219/00608 20130101; B01J 2219/0061
20130101; B01J 2219/00637 20130101; B82Y 30/00 20130101; B01J
2219/00722 20130101 |
Class at
Publication: |
435/006 ;
525/054.2; 536/024.3; 977/924; 977/754 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C08G 63/91 20060101
C08G063/91 |
Claims
1. A method for synthesis of a polynucleotide on a dendron-modified
surface of a substrate, wherein the dendron-modified surface is
obtained by chemically modifying with a dendron in which a
plurality of termini of the branched region of the dendron are
bound to the surface and a terminus of the linear region of the
dendron is functionalized.
2. The method according to claim 1, wherein the polynucleotide is
synthesized by reacting (a) at least one primer immobilized on the
dendron, with (b) a solution comprising polymerase, dNTP or NTP,
and template DNA or RNA.
3. The method according to claim 1, wherein the polynucleotide is
synthesized by reacting (a) template DNA or RNA immobilized on the
dendron, with (b) a solution comprising polymerase, dNTP or NTP,
and primers.
4. The method according to claim 1, wherein the polynucleotide is
synthesized by reacting (a) polymerase immobilized on the dendron,
with (b) a solution comprising dNTP or NTP, primers, and template
DNA or RNA.
5. The method according to claim 1, wherein the dendrons are spaced
at regular intervals between about 0.1 nm and about 100 nm among
the linear functionalized groups.
6. The method according to claim 1, wherein the terminus of the
branched region is functionalized with --COZ, --NHR, --OR', or
--PR''3, wherein Z is a leaving group, R is an alkyl, R' is alkyl,
aryl, or ether, and R'' is H, alkyl, or alkoxy.
7. The method according to claim 1, wherein the functional group is
--NH2, --OH, PH3, --COOH, --CHO or --SH.
8. The method according to claim 1, wherein the linear region
comprises a substituted or unsubstituted alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group.
9. The method according to claim 1, wherein the substrate is glass,
semiconductor, metal, plastics, silicone, silicate, metal alloy, or
synthetic organic metal.
10. The method according to claim 1, wherein the substrate is in a
form of a slide, a particle, a bead, a micro-well plate, or a
porous material.
11. The method according to claim 1, wherein the polynucleotide is
DNA, RNA, oligonucleotide, cDNA, nucleotide analog, or a
combination thereof.
12. The method according to claim 1, wherein the dendron-modified
surface is obtained by chemically modifying with the dendron after
treating the substrate with a silane compound having a hydroxyl
group.
13. The method according to claim 1, wherein the polynucleotide is
synthesized by a Klenow DNA polymerase I.
14. The method according to claim 1, wherein the synthesis of
polynucleotide is carried out by using RT-PCR or PCR method.
15. The method according to claim 14, wherein the PCR method
include a denaturing step, an annealing step, and an extension step
with Taq polymerase or a polymerase derived from Taq
polymerase.
16. A method of stably maintaining a polynucleotide immobilized on
a solid surface of a substrate under a thermal stress, wherein the
substrate is chemically modified with a dendron in which a
plurality of termini of the branched region of the dendron are
bound to the surface and a terminus of the linear region of the
dendron is functionalized.
17. The method of stably maintaining a polynucleotide according to
claim 16, wherein the thermal stress is a temperature of 60 to
100.quadrature..
18. The method of stably maintaining a polynucleotide according to
claim 16, wherein the thermal stress is long lasting.
19. The method of stably maintaining a polynucleotide according to
claim 16, wherein the thermal stress is put by repetitive heating
and cooling.
20. The method according to claim 16, wherein the substrate is
chemically modified with the dendron, after treating the substrate
with a silane compound having at least a hydroxyl group.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and the benefit of
Provisional Application No. 60/701,848 filed in the United States
Patent and Trademark Office on Jul. 22, 2005, the entire content of
which is incorporated hereinto by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for synthesis of a
polynucleotide on a dendron-modified surface of a substrate, and a
method for stably maintaining a polynucleotide immobilized on a
solid surface.
BACKGROUND OF THE INVENTION
[0003] The Polymerase Chain Reaction (PCR) technology has played an
important role in biotechnology, and no technique which could
replace the PCR technology has yet been developed. Accordingly, the
PCR technology will have an essential position in the field of
biotechnology in the future.
[0004] In addition to the development of the PCR technique, the
Reverse transcriptase-Polymerase Chain Reaction (RT-PCR) technique
which is a method of analyzing the gene expression based on the PCR
technique was invented. The RT-PCR has a higher sensitivity in
detection of a small amount of RNA molecule than a Northern blot
analysis, a dot blot analysis, and a nuclease protection method,
and is simpler than in situ hybridization. In particular, the
RT-PCT is very useful in analyzing various samples in very small
amounts, and therefore, the RT-PCR method is also widely used in
clinical diagnosis.
[0005] Based on these advantages of the PCR and RT-PCR methods,
many attempts have been made to combine the methods with high
throughput and highly parallel method such as microarray. However,
unlike the PCR or RT-PCR which is typically performed in a
solution, the reactions which happen on a solid surface has many
disadvantages to be overcome such as non-specific adsorption,
steric hindrance and electrostatic interaction between
biomolecules, etc. Specifically, when the high temperature
condition is required for PCR, the stability between the surface
and organic thin film introduced on the surface must be maintained
at a high temperature. However, the organic thin film cannot
maintain its stability in a buffer solution under the high
temperature when the organic thin film is introduced on a glass or
gold solid surface that is widely used for immobilizing
biomolecules.
SUMMARY OF THE INVENTION
[0006] The dendron-modified surface of a substrate according to the
present invention can make the biomolecules immobilized on the
solid surface to maintain a sufficient interval between the
biomolecules, thereby reducing undesirable steric hindrance and
static interaction between them. Thus, the present invention can
provide the conditions required for the PCR or RT-PCR to occur
under the same reaction conditions as those of the reactions in a
solution state.
[0007] In addition, an organic thin film coated on the
dendron-modified surface make it possible to minimize the
non-specific adsorption of biomolecules and to significantly
increase the thermal stability of biomolecules immobilized on the
organic thin film, thereby providing improved throughput/highly
parallel PCR and RT-PCR methods.
[0008] The present invention relates to a method for synthesis of
polynucleotide on a dendron-modified surface of substrate, where
the dendron-modified surface is obtained by chemically modifying a
dendron in which a plurality of termini of the branched region of
the dendron are bound to the surface, and a terminus of the linear
region of the dendron is functionalized.
[0009] In an embodiment of the present invention for synthesis of
polynucleotide on the dendron-modified surface of a substrate, the
polynucleotide is synthesized by reacting (a) at least a primer
immobilized on the dendron; with (b) a solution comprising
polymerase, dNTP or NTP, and template DNA or RNA.
[0010] In another embodiment of the present invention for synthesis
of polynucleotide on the dendron-modified surface of a substrate,
the polynucleotide is synthesized by reacting (a) template DNA or
RNA immobilized on the dendron; with (b) a solution comprising
polymerase, dNTP or NTP, and primers.
[0011] In further embodiment of the present invention for synthesis
of polynucleotide on the dendron-modified surface of a substrate,
the polynucleotide is synthesized by reacting (a) polymerase
immobilized on the dendron; with (b) a solution comprising dNTP or
NTP, primers and template DNA or RNA.
[0012] According to an embodiment of the present invention, the
synthesis of polynucleotide can be performed under a high
temperature or under the thermal cycles, wherein heating and
cooling are repeated.
[0013] In addition, the present invention also provides a method of
stably maintaining a polynucleotide immobilized on a solid surface
of a substrate under a thermal stress, wherein the substrate is
chemically modified with a dendron such that a plurality of termini
of the branched region of the dendron are bound to the surface, and
a terminus of the linear region of the dendron is functionalized.
In the method of stably maintaining a polynucleotide immobilized on
a solid surface of a substrate, the substrate is chemically
modified with the dendron, after treating the substrate with a
silane compound having a hydroxyl group.
BRIEF DESCRIPTION OF THE DRAWING
[0014] A more complete appreciation of the invention, and many of
the attendant advantages thereof, will be readily apparent as the
same becomes better understood by reference to the following
detailed description when considered in conjunction with the
accompanying drawing, wherein:
[0015] FIG. 1 is a schematic view showing each DNA chain extension
reaction on the dendron-modified surface: (A) The DNA chain
extension is performed by reacting the primers immobilized on the
dendron-modified surface with free template DNA under the presence
of free enzyme; (B) DNA chain extension is performed by reacting
template DNA immobilized on the dendron-modified surface with the
free primers under the presence of free enzyme; (C) DNA chain
extension is performed by reacting the free DNA template with the
free primers under the presence of enzyme immobilized on the
dendron-modified surface.
[0016] FIG. 2 is a schematic view showing DNA immobilized on the
dendron-modified surface, (a) the meso-spaced dendron surface of
solid support, and (b) a conventional surface of solid support.
[0017] FIG. 3 shows the thermal stability of the silanated slide
(A), and fluorescent image of the silanated slide (B), wherein the
numerical value under each image means the number of surface
treatment with PCR solution of high temperature.
[0018] FIG. 4 shows the thermal stability of Dendron/(ethylene
glycol(EG)/(3-glycidoxypropyl)methyldiethoxysilane (GPDES) slide
(A), and a fluorescent image of Dendron/EG/GPDES slide (B), wherein
the numerical value under each image means the number of surface
treatment with PCR solution of high temperature.
[0019] FIG. 5 represents the thermal stability of Dendron/TPU slide
(A), and a fluorescent image of Dendron/TPU slide (B), wherein the
numerical value under each image means the number of surface
treatment with PCR solution of high temperature.
[0020] FIG. 6 is a schematic view showing the DNA extension
reaction following the binding between the primer DNA and template
DNA.
[0021] FIG. 7 is a fluorescent image of DNA microarray obtained
from the chain extension by using Klenow DNA polymerase I: (A) Cy5
fluorescent image and (B) Cy3 fluorescent image.
[0022] FIG. 8 is a fluorescent image of DNA microarray obtained
from the chain extension by using Tag polymerase: (A) Cy5
fluorescent image and (B) Cy3 fluorescent image.
DETAILED DESCRIPTION
[0023] The present invention relates to a method of performing PCT
or RT-PCR on the dendron-modified surface, and more specifically to
a thermal stability of an organic thin film introduced on the
dendron-modified surface and biomolecules immobilized on the
organic thin film, and to a chain extension of PCR or RT-PCR.
[0024] According to most of the amplification methods, such as PCR,
RT-PCR, random priming method, and other similar DNA amplification
methods, DNA or RNA can be amplified through extension process.
However, most of the extension processes require enzymes having a
high optimal temperature such as Taq polymerase in order to reduce
the amplification time and to increase the amplification
efficiency. Thus, to carry out PCR, RT-PCR, random priming method,
and other similar DNA amplification methods on the solid surface,
an organic thin film introduced on the surface and biomolecules
immobilized on the organic thin film must be thermally stable. In
addition, the dendron-modified surface provides sufficient
intervals between the immobilized biomolecules, thereby allowing
the immobilized biomolecules to interact smoothly with other
biomolecules in the solution. Therefore, the polynucleotide
synthesis such as DNA or RNA synthesis, DNA or RNA chain extension,
PCR, RT-PCR, random priming nucleic acid synthesis, or the other
similar polynucleotide synthesis, chain extension, or any
amplification method can be carried out on the dendron-modified
surface successfully and efficiently.
[0025] The dendron materials and the preparation method of the thin
film on a solid surface is disclosed in US Publication No.
20050037413A1, the entire content of which is incorporated hereinto
by reference.
[0026] A dendron has a plurality of termini of the branched region
of the dendron which are bound to the surface, and a terminus of
the linear region of the dendron which is functionalized. The
dendrons are spaced at regular intervals between about 0.1 nm and
about 100 nm among the linear functionalized groups. In particular,
the macromolecules may be spaced at regular intervals of about 10
nm.
[0027] The terminus of the branched region may be functionalized
with --COZ, --NHR, --OR', or --PR''.sub.3, wherein Z may be a
leaving group, wherein R may be an alkyl, wherein R' may be alkyl,
aryl, or ether, and R'' may be H, alkyl, alkoxy, or O. In
particular, COZ may be ester, activated ester, acid halide,
activated amide, or CO-imiazoyl; R may be C.sub.1-C.sub.4 alkyl,
and R' may be C.sub.l-C.sub.4 alkyl. Further, in the above
described substrate, the polymer may be a dendron. Still further,
the linear region of the polymer may be comprised of a spacer
region. And the spacer region may be connected to the branched
region via a first functional group. Such first functional group
may be without limitation --NH.sub.2, --OH, --PH.sub.3, --COOH,
--CHO, or --SH. Still further, the spacer region may comprise a
linker region covalently bound to the first functional group.
[0028] In the substrate described above, the linker region may
comprise a substituted or unsubstituted alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group.
Still further, spacer region may comprise a second functional
group. The second functional group may include without limitation
--NH.sub.2, --OH, --PH.sub.3, --COOH, --CHO, or --SH. The second
functional group may be positioned at the terminus of the linear
region and a protecting group may be bound to the terminus of the
linear region. The protecting group may be acid labile or base
labile.
[0029] The surface materials on which dendron thin film can be
introduced are disclosed in US Publication No. 20050037413A1, the
entire content of which is incorporated hereinto by reference. Such
materials are used in the present invention.
[0030] In yet another embodiment of the invention, the substrate
described above may consist of semiconductor, synthetic organic
metal, synthetic semiconductor, metal, alloy, plastic, silicon,
silicate, glass, or ceramic. In particular, the substrate may be,
without limitation, a slide, particle, bead, micro-well plate, AFM
(atomic force measurement) cantilever or porous material. The
porous material may be a membrane, gelatin or hydrogel. And
particularly, the bead may be a controlled pore bead.
[0031] It has been known that an organic thin film introduced on a
glass surface by using the silane reaction is not stable in a
buffer solution at a high temperature. (Anal. Chem. 2004, 76,
1778-1787). Even though its unstability in a buffer solution at a
high temperature was confirmed again in this experiment, the
present inventors found that the thermal stability of the organic
thin film was dependent on the type of organic thin film.
[0032] Thus, the substrate is modified with the dendron. That is,
the dendron-modified surface of the substrate is obtained by
chemically modifying with the dendron, after treating the substrate
with a silane compound having a hydroxyl group. Examples of silane
compounds include (3-glycidoxypropyl) methyldiethoxysilane (GPDES)
and N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane
(TPU), but they are not limited thereto.
[0033] Herein, the term "polynucleotide synthesis" includes
polynucleotide synthesis, chain extension, and amplification. For
example, the term includes DNA or RNA polymerizaton such as DNA or
RNA synthesis, DNA or RNA chain extension, PCR, RT-PCR, random
priming nucleic acid synthesis, or the other similar polynucleotide
synthesis, chain extension, or any amplification method. The term,
"polynucleotide" means DNA, RNA, oligonucleotide, cDNA, nucleotide
analog or a combination thereof.
[0034] Any one of the enzymes used in standard PCR and RT-PCR,
including Tag DNA polymerase and the modified Tag DNA polymerase,
can be used for the present invention. In an embodiment of the
present invention, the PCR method is performed by a denaturing
step, an annealing step, and an extension step with Taq polymerase
or a polymerase derived from Taq polymerase.
[0035] Herein, the term, "reaction buffer solution" is referred to
a buffer solution used in amplification methods such as PCR,
RT-PCR, random priming method, and the other similar amplification
method. Examples of the buffer solution includes buffer solution 1
(50 mM KCl, 10 mM Tris-HCl, and 1.5 mM MgCl.sub.2), buffer solution
2 (10 mM Tris-HCl, 40 mM KCl, 1.5 mM MgCl.sub.2), and buffer
solution 3 (50 mm Tris-HCl, 10 mM MgCl.sub.2, 1 mM DTT, 50 .mu.g/ml
BSA), but are not limited thereto.
[0036] The present invention uses the method of immobilizing the
biomolecules disclosed in US publication No. 20050037413A 1.
[0037] The present invention relates to a method for synthesis of
polynucleotide on dendron-modified surface of substrate, where the
dendron-modified surface is obtained by chemically modifying with a
dendron in which a plurality of termini of the branched region of
the dendron are bound to the surface, and a terminus of the linear
region of the dendron is functionalized.
[0038] The polynucleotide synthesis can be performed using the
following three methods for example.
[0039] In an embodiment of the present invention for the synthesis
of polynucleotide on the dendron-modified surface of substrate, the
polynucleotide is synthesized by reacting (a) at least a primer
immobilized on the dendron; with (b) a solution comprising
polymerase, dNTP or NTP, and template DNA or RNA.
[0040] In another embodiment of the present invention for the
synthesis of polynucleotide on the dendron-modified surface of
substrate, the polynucleotide is synthesized by reacting (a)
template DNA or RNA immobilized on the dendron; with (b) a solution
comprising polymerase, dNTP or NTP, and primers.
[0041] In further embodiment of the present invention for the
synthesis of polynucleotide on the dendron-modified surface of
substrate, the polynucleotide is synthesized by reacting (a)
polymerase immobilized on the dendron; with (b) a solution
comprising dNTP or NTP, primers and template DNA or RNA.
[0042] In yet another embodiment of the present invention, the
synthesis of polynucleotide can be performed under a high
temperature, or under the thermal cycles that heating and cooling
are repeated. The reaction temperature of polynucleotide synthesis
can be different depending on the enzymes and synthesizing methods.
For example, the reaction temperature of polynucleotide synthesis
is 30.degree. C. to 100.degree. C., preferably 35.degree. C. to
100.degree. C, and more preferably 70.degree. C. to 98.degree.
C.
[0043] In addition, the present invention provides a method of
stably maintaining a polynucleotide immobilized on a solid surface
of a substrate under a thermal stress. The thermal stress can
result from repetitive heating and cooling and can be long lasting.
For example, the thermal stress can be a high temperature, such as
a temperature in the range of 60 to 100.quadrature., and preferably
70 to 100.quadrature.. The substrate is chemically modified with a
dendron such that a plurality of termini of the branched region of
the dendron are bound to the surface, and a terminus of the linear
region of the dendron is functionalized. In the method of stably
maintaining a polynucleotide immobilized on a solid surface of a
substrate, the substrate is chemically modified with the dendron,
after treating the substrate with a silane compound having a
hydroxyl group. The dendron, substrate, and biomolecules as
described above can be used.
[0044] The following Example 1 shows that the dendron-modified
surface had sufficient thermal stability in a reaction buffer
solution. In the Example 2, the chain extension could be performed
efficiently and successfully under a high temperature and also
under a low temperature. It confirmed that the dendron-modified
surface was suitable for PCR, RT-PCR, random priming and the other
similar amplification methods.
[0045] The present invention is further explained in more detail
with reference to the following examples. The scope of the present
invention, however, is not limited to the following examples.
EXAMPLE 1
Preparation of Dendron-Modified Substrate
[0046] The two types of the modification (9-acid/GPDES substrate
and 9-acid/TPU substrate) were employed for the substrate by using
the two silane agents GPDES and TPU.
Example 1.1
[0047] Materials
[0048] The silane coupling reagents,
(3-glycidoxypropyl)methyldiethoxysilane (GPDES) and
N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide urethane (TPU) was
purchased from Gelest Inc. and all other chemicals were of reagent
grade from Sigma-Aldrich. Reaction solvents for the silylation are
anhydrous ones in Sure/Seal bottles from Aldrich. All washing
solvents for the substrates are of HPLC grade from Mallinckrodt
Laboratory Chemicals. Glass slides (2.5.times.7.5 cm) were
purchased from Corning Co. Ultrapure water (18 M .OMEGA./cm) was
obtained from a Milli-Q purification system (Millipore).
Example 1.2
[0049] Cleaning the Substrates
[0050] Glass slide as a substrate was immersed into Piranha
solution (conc. H.sub.2SO.sub.4:30% H.sub.2O.sub.2=7:3 (v/v)) and a
reaction bottle containing the solution and the substrates was
sonicated for an hour. The plates were washed and rinsed thoroughly
with a copious amount of deionized water after the sonication. The
clean substrates were dried in a vacuum chamber (30-40 mTorr) for
the steps to be followed.
Example 1.3
[0051] Preparing the Hydroxylated Substrates
[0052] The above clean substrates were soaked in 180 ml toluene
solution with 1.0 ml (3-glycidoxypropyl)methyldiethoxysilane
(GPDES) for 4 hours. After the self-assembly, the substrates were
washed with toluene briefly, placed in an oven, and heated at
110.degree. C. for 30 minutes. The plates were sonicated in
toluene, toluene-ethanol (1:1 (v/v)), and ethanol in a sequential
manner for 3 min at each washing step. The washed plates were dried
in a vacuum chamber (30-40 mTorr). GPDES-modified substrates were
soaked in a neat ethylene glycol (EG) solution at 80-100.degree. C.
for 8 h. After cooling, the substrates were sonicated in D.I water
and ethanol in a sequential manner each for 3 min. The washed
plates were dried in a vacuum chamber (30-40 mTorr).
[0053] Clean slide glass was immersed into anhydrous toluene (20
mL) containing N-(3-(triethoxysilyl)propyl)-O-polyethyleneoxide
urethane (TPU) as a silane coupling agent (0.20 mL) under nitrogen
atmosphere, and placed in the solution for 6 h. After silylation,
the substrates were washed with toluene, baked for 30 min at
110.degree. C. The substrates were immersed in toluene,
toluene-ethanol (1:1 (v/v)), and ethanol in a sequential manner,
and they were sonicated for 3 min in each washing solution. The
substrates were rinsed thoroughly with toluene and methanol in a
sequential manner. Finally the substrates were dried under vacuum
(30-40 mTorr).
Example 1.4
[0054] Preparing the Dendron-Modified Substrates
[0055] The above hydroxylated substrates were immersed into a
methylene chloride solution dissolving (9-anthrylmethyl
N-({[tris({2-[({tris[(2-carboxyethoxy)methyl]methyl}amino)carbonyl]ethoxy-
}methyl)methyl]amino}carbonyl)propylcarbamate) (or 9-acid) (0.5 mM)
and a coupling agent,
1-[-3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDC) or 1,3-dicyclohexylcarbodiimide (DCC) (5 mM) in the presence
of 4-dimethylaminopyridine (DMAP) (4 mM). After 3 days at room
temperature, the plates were sonicated in methanol, water, and
ethanol in the respective sequence each for 3 minutes. The washed
plates were dried in a vacuum chamber (30-40 mTorr) for the step to
be followed.
Example 1.5
[0056] Preparing the NHS-Modified Substrates
[0057] The dendron-modified substrates were immersed into a
methylene chloride solution with 0.1 M trifluoroacetic acid (TFA).
After 3 hours, they were again soaked in a methylene chloride
solution with 1% (v/v) triethylamine(TEA) for 10 minutes. The
plates were sonicated in methylene chloride and ethanol each for 3
minutes. After being dried in a vacuum chamber, the deprotected
substrates were incubated in acetonitrile solution with
di(N-succinimidyl)carbonate (DSC) (25 mM) and DIPEA (1.0 mM). After
4 hours of reaction under nitrogen atmosphere, the plates were
placed in a stirred dimethylformamide solution for 30 min and then
were washed briefly with methanol. The washed plates were dried in
a vacuum chamber (30-40 mTorr) for the step to be followed.
EXAMPLE 2
Thermal Stability of the Immobilized DNAs on Dendron-Modified
Surface
[0058] To test the thermal stability of the biomolecules on several
different surfaces, Silanted slide, Dendron/EG/GPDES slide, and
Dendron/TPU slide were used in this example. This example is to
compare how DNA molecules immobilized on the dendron-modified
surface of the present invention and on the aminosilane treated
surface used in the conventional art were maintained stably in
buffer solution at a high temperature. The Silanted slide (TeleChem
International, Inc) which was treated with aminosilane was used as
a comparative example.
[0059] Dendron/EG/GPDES slide, and Dendron/TPU slide were the same
as those of Example 1.
[0060] The oligonucleotides used in this example included an amino
group at 3' end and Cy3 dye at 5' end as follows:
[0061] 5'Cy3-TTT TTT TTT T-NH.sub.2-3' (SEQ ID NO: 1)
[0062] A PCR buffer solution (50 mM KCl, 10 mM Tris-HCl, and 1.5 mM
MgCl.sub.2 (pH 7.4)) was used as the buffer solution for measuring
the thermal stability.
[0063] The oligonucleotides including a fluorescent dye were
spotted on the dendron-modified surface of Example 1 with a
microarrayer and the surface was incubated for a sufficient time to
allow the oligonucleotides to be immobilized on the surface.
Unreacted oligonucleotides were removed by rinsing with a washing
buffer. The glass slide was dried, and then the fluorescence signal
of the immobilized oligonucleotides was measured using a laser
fluorescent scanner.
[0064] The glass slide showing the fluorescent signal was immersed
in a PCR buffer solution at a temperature of 92-98.quadrature. for
5 minutes, washed with deionized water and dried before the
fluorescence signal of the immobilized oligonuleotides was measured
using a laser fluorescent scanner. Again, the glass slide showing
the fluorescent signal was immersed in a PCR buffer solution at a
temperature of 92-98.quadrature. for 5 minutes, washed with
deionized water and dried before the fluorescence signal of the
immobilized oligonuleotides was measured using a laser fluorescent
scanner. The repetitive experiments as described above were carried
out and then the intensity of fluorescent signal was analyzed for
different repetition numbers.
[0065] It has been known that an organic thin film introduced on a
glass surface by using the silane reaction is not stable in a
buffer solution at a high temperature. (Anal. Chem. 2004, 76,
1778-1787). Even though the unstability was confirmed here again,
the present inventors found that the thermal stability of the
organic thin film depends on the type of organic thin film. The
Silanated slide used as a comparative example showed steep decrease
of the fluorescent intensity as the repetition number increased
(FIG. 3A and FIG. 3B). However, although the dendron-modified
surface showed the decreased intensity of the fluorescent signal,
the amount of decrease was significantly smaller compared to that
of Silanated slide (FIG. 4A and FIG. 4B).
[0066] In addition, in the preparation method of the
dendron-modified solid surface, the solid surface treated with TPU
(N-(3-(triethoxysilyl)propyl)-o-polyethylene oxide urethane) (FIG.
5A and FIG. 5B) was shown to be thermally more stable than those
treated with GPDES ((3-glycidoxypropyl)methyldiethoxysilane) and
ethylene glycol (FIG. 4A and FIG. 4B). It suggested that the TPU
organic thin film block off the salts in buffer solution
approaching the glass slide surface efficiently, and minimized the
damage of Si--O bond.
EXAMPLE 3
Thermal Cycle Reaction With Enzymes on Dendron-Modified Surface
[0067] Based on the thermal stability test in Example 2, PCR,
RT-PCR or the other similar thermal cycles could be performed on
the dendron-modified surface by carrying out a general PCR and
RT-PCR procedures on the surface.
[0068] As a comparative example, the Silanted slide treated with
aminosilane (TeleChem International, Inc.) was used in this
example.
[0069] Dendron/EG/GPDES slide, and Dendron/TPU slide were the same
as those of Example 1.
[0070] The Tag DNA polymerase generally used in PCR was used in
this example. The buffer solution for Tag DNA polymerase included
40 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl.sub.2, but could be
different according to the enzymes used. The buffer solution for
DNA polymerase can be adjusted depending on the enzyme used.
[0071] The oligonucleotides used in this example included an amino
group at 3' end and Cy3 dye at 5' end as follows:
[0072] 5'-Cy3-ACA AGC ACA GTT AGG-NH.sub.2-3' (SEQ ID NO: 2)
[0073] The oligonucleotides including a fluorescent dye were
spotted on the dendron-modified surface of Example 1 with a
microarrayer and the surface was incubated in a sufficient time to
allow the oligonucleotides to be immobilized on the surface.
Unreacted oligonucleotides were removed by rinsing with a washing
buffer. The glass slide was dried, and then the fluorescence signal
of the immobilized oligonucleotides was measured using a laser
fluorescent scanner.
[0074] The glass slide was immersed in a buffer solution containing
100 .mu.M dATP, 100 .mu.M dTTP, 100 .mu.M dCTP, 100 .mu.M dGTP, and
Tag DNA polymerase, and heated at 94.degree. C. for 2 minutes.
Then, the heating cycle which was at 94.degree. C. for 20 seconds,
at 60.degree. C. for 20 seconds, and at 72.degree. C. for 20
seconds was repeated at 20 cycles sequentially, and then was at
72.degree. C. for 7 minutes for the last step. The glass slide was
washed with deionized water, dried, and then the fluorescence
signal of the immobilized oligonucleotides was measured using a
laser fluorescent scanner to compare the intensities of fluorescent
signals in the samples obtained before and after PCR.
[0075] As a result, the comparative example of Silanated slide
showed 20,000 of fluorescent intensity before PCR but showed a
steep decrease of the intensity to 2,000 after PCR. On the other
hand, dendron/TPU slide showed much smaller decrease of the
fluorescent intensity from 15,000 before PCR to 11,000. This result
was consistent with that of thermal stability obtained in Example
2, and represented that the dendron-modified surface provided
organic thin film with more stability than the general silanated
slide. Thus, the result confirmed that PCR, RT-PCR and other
thermal cycle procedures similar to PCR or RT-PCR could be carried
out efficiently.
EXAMPLE 4
4.1. DNA Extension by Enzymes on Dendron-Modified Surface
[0076] The PCR and RT-PCR methods include a denaturing step, an
annealing step, and an extension step. This Example was performed
to measure the efficiency of the extension step on the solid
surface.
[0077] The dendron-modified surface used in this Example was the
same as that of Example 1, and the oligonucleotides to be
immobilized on the surface were as follows. TABLE-US-00001 TABLE 1
NAME SEQUENCE(5' to 3') SEQ ID NO Primer 1
5'-NH2-gatcaccagcggcatcgag - 3' 3 Primer 2
5'-NH2-gatcaccaccggcatcgag -3' 4 Primer 3
5'-NH2-cgatcaccaacggcatcgag -3' 5 Primer 4
5'-NH2-cgatcaccatcggcatcgag -3' 6 Primer 5 5'-NH2-atcacccgcggcatcga
-3' 7
[0078] The oligonucleotide as described in Table 1 are markers to
detect katG gene of Mycobacterium tuberculosis, and particularly
mutated kat G gene in codon 315. Primer 1 is designed for detect
wild type Mycobacterium tuberculosis, and Primers 2 to 5 are
designed for detecting a mutant type. Primers 1 to 5 had NH2 group
on their 5'-end. The template DNA which the oligonucleotides detect
is isolated from Mycobacterium tuberculosis, the 315
codon-containing gene fragment of isolated whole gene is only
amplified with PCR where the dCTP-Cy5 was added to be labeled. The
template DNA has a length of about 200 base pairs. Klenow DNA
polymerase I and Tag polymerase I were used at their optimal
temperature of 37.degree. C. and 72.degree. C., respectively.
[0079] FIG. 6 is a schematic view showing the DNA extension
reaction following the binding between the primer DNA and the
template DNA. Firstly, the primers including a fluorescent dye were
spotted on the dendron-modified surface with a microarrayer, and
the surface was incubated in a sufficient time for the
oligonucleotides to immobilize on the surface. Unreacted primers
were removed by rinsing with a washing buffer. The glass slide was
then dried. The obtained DNA microarray was hybridized the template
DNA at a specific temperature for 2 hours, and the unhybridized
template DNAs were removed by washing with a buffer solution, and
then were dried.
4.2. DNA Extension on Dendron-Modified Surface by Using Klenow DNA
Polymerase I
[0080] The obtained DNA microarray was incubated in Klenow DNA
polymerase I reaction buffer solution at 37.degree. C. for 10
minutes in order to sufficiently soak DNA microarray on the slide,
and then incubated with addition of a buffer solution including
Klenow DNA polymerase I, 100 .mu.M dATP, 100 .mu.M dTTP, 100 .mu.M
dGTP, 50 .mu.M dCTP, and 50 .mu.M dCTP-Cy3 at 37.degree. C. for 30
minutes. The DNA microarray was washed with PBS, and dried before
the fluorescent signal of DNA microarray was measured.
[0081] FIG. 7 is a fluorescent image of DNA microarray obtained
from the chain extension by using Klenow DNA polymerase I: (A) Cy5
fluorescent image which showed the fluorescent signal of the
Cy5-labeled Template DNA hybridized with primers, and (B) Cy3
fluorescent image which showed the fluorescent signal of the
dCTP-Cy3 incorporated into the amplified DNA produced from DNA
extension on the dendron-modified surface. The result of FIG. 7A
represented that the hybridization between the primers and template
DNA on the dendron-modified surface occurred with high selectivity.
The result of FIG. 7B confirmed that the DNA extension on the solid
surface by using Klenow DNA polymerase I was performed
successfully. As shown in FIGS. 7A and 7B, the hybridization and
polynucleotide synthesis performed by using Primer 1 which did not
include a mismatched base, and Primer 2 to 5 which included 1
mismatched base pair showed that only Primer 1 provided complete
hybridization with the template DNA and the DNA extension. Such
result suggested that the dendron-modified surface of the present
invention have high selectivity to matching and mismatching.
4.3. DNA Extension on Dendron-Modified Surface by Using Tag DNA
Polymerase
[0082] The DNA microarray after DNA hybridization with template DNA
was immersed in Tag DNA polymerase reaction buffer solution, and
incubated at 72.degree. C. for 10 minutes to allow the upper side
of the DNA microarray on the slide to be wet sufficiently. The DNA
microarray was immersed in a buffer solution containing Tag DNA
polymerase, 100 .mu.M dATP, 100 .mu.M dTTP, 100 .mu.M dGTP, 50
.mu.M dCTP, and 50 .mu.M dCTP-Cy3, and was incubated at 72.degree.
C. for 5 minutes. Then, the microarray was washed with PBS, and
dried before its fluorescence was measured.
[0083] FIG. 8 is a fluorescent image of DNA microarray obtained
from the chain extension by using Taq polymerase: (A) Cy5
fluorescent image which showed the fluorescent signal of the
Cy5-labeled Template DNA hybridized with primers, and (B) Cy3
fluorescent image which showed the fluorescent signal of the
dCTP-Cy3 incorporated into the amplified DNA produced from DNA
extension on the dendron-modified surface.
[0084] The result of FIG. 8A represented that the hybridization
between the primers and the template DNA on the dendron-modified
surface occurred with high selectivity. The result of FIG. 8B
confirmed that the DNA extension on the solid surface by using Taq
polymerase was performed successfully.
[0085] As shown in FIG. 8A and FIG. 8B, in the case of the chain
extension by using Primer 1 which does not include a mismatched
base, and Primers 2 to 5 which include 1 bp mismatched, the
hybridization and polynucleotide synthesis results showed that only
Primer 1 completely hybridized template DNA, and thus provided the
extension reaction. Thus, the dendron-modified surface showed high
selectivity to the base pair matching and mismatching.
[0086] While the present invention has been described in detail
with reference to the preferred embodiments, those skilled in the
art will appreciate that various modifications and substitutions
can be made thereto without departing from the spirit and scope of
the present invention as set forth in the appended claims.
Sequence CWU 1
1
7 1 10 DNA Artificial Sequence DNA used in Example 2 1 tttttttttt
10 2 15 DNA Artificial Sequence DNA used in Example 3 2 acaagcacag
ttagg 15 3 19 DNA Artificial Sequence Primer 1 3 gatcaccagc
ggcatcgag 19 4 19 DNA Artificial Sequence Primer 2 4 gatcaccacc
ggcatcgag 19 5 20 DNA Artificial Sequence Primer 3 5 cgatcaccaa
cggcatcgag 20 6 20 DNA Artificial Sequence Primer 4 6 cgatcaccat
cggcatcgag 20 7 17 DNA Artificial Sequence Primer 5 7 atcacccgcg
gcatcga 17
* * * * *